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J Biol Chem, Vol. 274, Issue 30, 21003-21010, July 23, 1999

Fyn and JAK2 Mediate Ras Activation by Reactive Oxygen Species^*

Jun-ichi Abe and Bradford C. Berk

From the Center for Cardiovascular Research, University of Rochester, Rochester, New York 14642

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Reactive oxygen species (ROS) activate Ras and the extracellular signal-regulated kinase (ERK) cascade. Because JAK2 is a critical mediator for Ras/Raf/ERK activation by several hormones, we examined the role of JAK2 in ROS signal events. H₂O₂ stimulated JAK2 activity in fibroblasts with peak at 2-5 min. To determine the specific role of Src and Fyn as mediators of JAK2 activation and its downstream events, we used fibroblasts derived from transgenic mice deficient in Src (Src/) or Fyn (Fyn/). H₂O₂-stimulated JAK2 activity was completely inhibited in Fyn/ cells. Shc tyrosine phosphorylation and Ras activation by H₂O₂ were also significantly reduced in Fyn/ cells, but not altered in Src/ cells. Activation of JAK2 was restored when Fyn/ cells were transfected with B-Fyn but not with Src. Inhibiting JAK2 activity with the specific inhibitor AG-490 prevented H₂O₂ stimulated Shc and Ras activation. H₂O₂-mediated ERK1/2 activation in Fyn/ cells and AG-490 treated cells was completely inhibited at an early time (5 min), but not at late times (20-40 min) after stimulation. These results define a new redox-sensitive pathway for Ras activation and rapid ERK1/2 activation, which is mediated by Fyn and JAK2.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Reduction-oxidation (redox) reactions that generate reactive oxygen species (ROS),¹ including H₂O₂, O₂, and OH, have been identified as important chemical mediators that regulate signal transduction. Because increased ROS may be a risk factor for cardiovascular events such as unstable angina, myocardial infarction, and sudden death, understanding the biological processes that generate ROS and the intracellular signals elicited by ROS will be useful to gain insights into the pathogenesis of these diseases (1-4). Recently, it has been shown that ROS stimulate intracellular signal events similar to those activated by growth factors including stimulating kinases and small G proteins such as c-Src, Ras, and ERK1/2 (5, 6). Lander et al. (7) have reported that p21 Ras is a direct target of ROS and thus may be responsible for sensing redox status. In addition, Guyton et al. (8) have shown that H₂O₂-stimulated activation of ERK2 was abolished in PC12 cells by expression of dominant-negative Ras. These findings suggest that p21Ras may be an important mediator of ROS function.

Previous studies have shown that c-Src is involved in signal events stimulated by ROS (5, 9). It has been reported that Src family kinases and Ras are critical for ERK1/2 activation by H₂O₂ (10, 11). The predominant pathway for ERK1/2 activation by angiotensin II and growth factors has also been proposed to involve Src-Ras-Raf-MEK1-ERK1/2 (12, 13). However, it is not known how Src kinases mediate Ras activation by ROS. Several groups have suggested that JAK2 activates the Ras/Raf/ERK-signaling pathway and is required for the proliferative response initiated by many cytokines (14). Han et al. (15) have reported that JAK2 is required to couple growth hormone receptor to pathways involving Shc. Shc is thought to function as an adapter molecule to recruit Grb2·Sos complexes to the activated receptor, which promotes formation of Ras-GTP (16). In Src-transformed cells, JAK1 and JAK2 are constitutively tyrosine phosphorylated (17) suggesting that H₂O₂-mediated Shc/Ras activation may be regulated by Src family kinases and JAK2.

To determine the role of Src family kinases and JAK2 in H₂O₂-mediated Shc/Ras activation, we used specific JAK2 inhibitors and cells derived from animals deficient in c-Src and Fyn. We show here that activation of JAK2 by H₂O₂ is positively regulated by Fyn, but not by c-Src. Furthermore, we demonstrate that both Fyn and JAK2 are required for H₂O₂-mediated Shc tyrosine phosphorylation and activation of Ras. Thus, the Fyn-JAK2-Shc-Ras-signaling pathway described here may represent a new redox-sensitive mechanism.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Cell Lines, Culture, and Transfection-- Fibroblasts deficient in c-Src (Src/) or Fyn (Fyn/) were isolated from mouse embryo fibroblasts homozygous for disruption of the Src and Fyn genes, immortalized with large T antigen (18). Cells were kindly provided by Sheila M. Thomas, Fred Hutchinson Cancer Center, Seattle, WA. Fibroblasts were maintained in Dulbecco's modified Eagle's medium supplemented with 10% calf serum as described previously (18). Cells at 70-80% confluence in 100-mm dishes were growth arrested by incubation in RPMI 1640 for 24 h before use. pME18S mammalian cell expression vectors encoding B-Fyn or Src cDNA were kindly provided by Drs. Hisashi Umemori and Tadashi Yamamoto, University of Tokyo. For transient expression experiments, cells were transfected 1 day after replating by LipofectAMINE method as described previously (19). After 48 h of incubation, cells were harvested for experiments.

Immunoprecipitation and Western Blot Analysis-- After treatment, the cells were washed with phosphate-buffered saline, harvested in 0.5 ml of lysis buffer (50 mM sodium pyrophosphate, 50 mM NaF, 50 mM NaCl, 5 mM EDTA, 5 mM EGTA, 100 µM Na₃VO₄, 10 mM HEPES, pH 7.4, 0.1% Triton X-100, 500 µM phenylmethanesulfonyl fluoride, and 10 µg/ml leupeptin), and flash-frozen on a dry ice/ethanol bath. After allowing the cells to thaw, cells were scraped off the dish and centrifuged at 14,000 × g (4 °C for 30 min), and protein concentration was determined using the Bradford protein assay (Bio-Rad). For immunoprecipitation, cell lysates were incubated with rabbit anti-JAK2 or Shc antibody (Upstate Biotechnology Inc.) for 3 h at 4 °C and then incubated with 20 µl of protein A-Sepharose CL-4B (Amersham Pharmacia Biotech) for 1 h on a roller system at 4 °C. The beads were washed 2 times with 1 ml of lysis buffer, 2 times with 1 ml of LiCl wash buffer (500 mM LiCl, 100 mM Tris-Cl, pH 7.6, 0.1% Triton X-100, 1 mM dithiothreitol) and 2 times in 1 ml of washing buffer (HEPES 20 mM, pH 7.2, 2 mM EGTA, 10 mM MgCl₂, 1 mM dithiothreitol, 0.1% Triton X-100). For Western blot analysis, cell lysates or immunoprecipitates were subjected to SDS-polyacrylamide gel electrophoresis, and proteins were transferred to nitrocellulose membranes (Hybond^TM-ECL, Amersham) as described previously (20) The membrane was blocked for 1 h at room temperature with a commercial blocking buffer from Life Technologies, Inc. The blots were then incubated for 4 h at room temperature with the anti-phosphotyrosine (4G10, U.B.I.), JAK2, Shc antibody, followed by incubation for 1 h with a secondary antibody (horseradish peroxidase conjugated). For ERK1/2 activation, the blots were incubated 12 h with anti-phospho-specific ERK1/2 (New England Biolabs) or nonspecific ERK1 and ERK2 antibodies (Santa Cruz Biotechnologies, Inc.). Immunoreactive bands were visualized using enhanced chemiluminescence (Amersham International, Uppala, Sweden).

Activated p21 Ras Affinity Precipitation Assay-- The expression vector encoding the fusion protein GST-Raf binding domain (RBD) was obtained by ligation of cDNA encoding the first 149 amino acids of Raf-1 into the SmaI site of the pGEX 2T vector (Amersham Pharmacia Biotech). GST-RBD expression was induced in transformed bacteria with 1 mM isopropyl -D-thiogalactoside for 1 h, after which time bacteria were harvested and lysed by sonication. The GST-RBD fusion protein was then purified on glutathione-Sepharose beads. Affinity precipitation of activated p21 Ras was performed as described previously (21, 22). Briefly, lysates were incubated on a rocker plate at 4 °C for 1 h with GST fusion protein bound to glutathione-Sepharose beads. Then, the supernatant containing equal amounts of protein after centrifugation was incubated with 50-60 µg of GST-RBD bound to beads at 4 °C for 4 h. The beads were then extensively washed with 20 mM HEPES, pH 7.5, 120 mM NaCl, 10% glycerol, 0.5% Triton X-100, 2 mM EDTA, 10 µg/ml leupeptin, and 10 µg/ml aprotinin. The eluted proteins were resolved on 14.0% polyacrylamide gel. Coomassie Brilliant Blue was used to stain the fusion protein in the gel (molecular mass 42 kDa). Bound p21Ras was quantified by Western blot analysis as described above.

Materials-- All materials were from Sigma except where indicated. H₂O₂ was from Fisher Scientific and AG-490 was from Calbiochem.

Statistical Analysis-- Data are reported as mean ± S.D. Statistical analysis was performed with the StatView 4.0 package (ABACUS Concepts, Berkeley, CA). Differences were analyzed with unpaired two-tailed Student's t test, or Welch's t test as appropriate.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

H2O₂ Stimulates JAK2 Kinase Activity in Fibroblasts-- JAK2 and Src have been suggested to be upstream of ERK1/2 in signal transduction cascades (13, 23, 24) and are likely candidates to mediate ROS signal transduction. To determine whether JAK2 was activated by exposure to 1 mM H₂O₂, cell lysates were immunoprecipitated with anti-JAK2 antibody, and Western blotting performed with anti-phosphotyrosine antibody (4G10). JAK2 was rapidly tyrosine phosphorylated, with increased phosphorylation within 30 s after the stimulation by H₂O₂ (Fig. 1, top). Peak tyrosine phosphorylation (9.56 ± 2.70-fold increase) occurred at 5 min and was sustained for 60 min (Fig. 1, bottom).

View larger version (57K): [in this window] [in a new window]   Fig. 1.   JAK2 is activated by H2O2 rapidly. Mouse fibroblasts were growth arrested for 24 h and stimulated with 1 mM H2O2 for the indicated times. Cells were harvested in lysis buffer, and proteins were immunoprecipitated with JAK2 antibody; Western blot analysis was performed with anti-phosphotyrosine antibody (4G10) (top) or anti-JAK2 antibody (bottom). No difference in the amount of JAK2 was observed in lysates from any of the cell lines by Western blot analysis with anti-JAK2.

H₂O₂ Stimulates JAK2 Tyrosine Phosphorylation via Fyn-dependent, Src-independent Mechanism-- To determine the role of Src family kinases in JAK2 activation by H₂O₂, we used cells derived from mice deficient in Src or Fyn (18). There was no immunoreactive c-Src in Src/ cells, whereas immunoreactive Fyn was expressed to the same extent as in wild type cells (Fig. 2A). Likewise, there was no immunoreactive Fyn in Fyn/ cells, although there was no change in expression of c-Src in Fyn/ cells compared with wild type cells (Fig. 2A). H₂O₂ stimulated JAK2 tyrosine phosphorylation in wild type fibroblasts, which was maximal at 5 min (Fig. 1). In Src/ fibroblasts, JAK2 tyrosine phosphorylation increased with maximum at 5 min (9.30 ± 2.34-fold increase) after H₂O₂ stimulation (Fig. 2B, top). In contrast, in Fyn/ fibroblasts, H₂O₂ failed to stimulate JAK2 tyrosine phosphorylation at any time (Fig. 2B, bottom). These results indicate that H₂O₂-mediated activation of JAK2 is dependent on Fyn, but not c-Src in fibroblasts.

View larger version (33K): [in this window] [in a new window]   Fig. 2.   H2O2 activation of JAK2 is inhibited in Fyn/ cells, but not in c-Src/ cells. A, wild type mouse fibroblast (WT), Src/, and Fyn/ cells were harvested, and Western blot analysis was performed on whole cell lysates using anti-Src antibody (left panel) and anti-Fyn antibody (right panel). B, cells were stimulated for the indicated times with 1 mM H2O2, and cell lysates were incubated with JAK2 antibody, and immunoprecipitates from each were analyzed by anti-phosphotyrosine and anti-JAK2 Western blotting. No difference in the amount of JAK2 was observed in lysates from any of the cell lines by Western blot analysis with anti-JAK2. C, densitometric analysis of JAK2 tyrosine phosphorylation. Results were normalized by arbitrarily setting the densitometry of control cells (time = 0) to 1.0 (shown is mean ± S.D., n = 3).

H₂O₂ Stimulates Shc Tyrosine Phosphorylation via a Fyn- and JAK2-dependent Mechanism-- Previously, Rao et al. (25) showed that the SH2-containing adapter molecule, Shc, became phosphorylated on tyrosine following treatment of cells with H₂O₂. Shc is thought to function as an adapter molecule to recruit Grb2-Sos complexes to activated receptors (16). In the case of erythropoetin, Shc appears to associate directly with JAK2 following erythropoetin treatment (26). Therefore, we investigated the role of JAK2 tyrosine kinase in H₂O₂-stimulated Shc phosphorylation. We first investigated the effect of AG-490, a specific JAK2 inhibitor (24, 27). Treatment with AG-490 for 16 h caused a concentration-dependent inhibition of H₂O₂-mediated JAK2 activation with an approximate IC₅₀ value of 30 µM (Fig. 3, A and B). In contrast, p130CAS tyrosine phosphorylation induced by H₂O₂ was unaffected by AG-490 treatment (Fig. 3C). Treatment with AG-490 also did not prevent H₂O₂-mediated tyrosine phosphorylation of Pyk2 and FAK (data not shown). Combined with previous data (27), these results strongly support the hypothesis that JAK2 is a specific target for AG-490 action in fibroblasts.

View larger version (35K): [in this window] [in a new window]   Fig. 3.   AG-490 inhibits JAK2 but not p130Cas tyrosine phosphorylation in a concentration-dependent manner in fibroblasts. Growth arrested fibroblasts were pretreated with Me2SO or the indicated concentrations of AG-490 for 16 h. Cell lysates were incubated with JAK2 (A) or p130Cas (B) antibody, and immunoprecipitates from each were analyzed by anti-phosphotyrosine (top) and anti-JAK2 (A) or p130Cas (B) Western blotting (bottom). No difference in the amount of JAK2 and p130Cas was observed in lysates from any of the cell lines by Western blot analysis with anti-JAK2 (A) or anti-p130Cas (C) (bottom). B, densitometric analysis of JAK2 tyrosine phosphorylation. Results were normalized by arbitrarily setting the densitometry of control cells (time = 0) to 1.0 (shown is mean ± S.D., n = 3).

Next we determined whether AG-490 inhibited H₂O₂-mediated Shc tyrosine phosphorylation. H₂O₂ stimulated Shc tyrosine phosphorylation within 1 min with maximum at 5 min (4.65 ± 0.90-fold increase) (Fig. 4). No difference in Shc protein expression was observed in lysates from control and H₂O₂-stimulated cells as determined by immunoprecipitation and Western blot analysis with anti-Shc antibody (Fig. 4, A and B). We found that JAK2 inhibition with 60 µM AG-490 decreased H₂O₂-induced Shc tyrosine phosphorylation by >90% (Fig. 5, A and B).

View larger version (55K): [in this window] [in a new window]   Fig. 4.   H2O2 stimulates Shc tyrosine phosphorylation rapidly. Mouse fibroblasts were growth arrested for 24 h and stimulated with 1 mM H2O2 for the indicated times. Cells were harvested in lysis buffer, and proteins were immunoprecipitated with Shc antibody; Western blot analysis was performed with anti-phosphotyrosine antibody (4G10) (top) or anti-Shc antibody (bottom). No difference in the amount of Shc was observed in lysates from any of the cell lines by Western blot analysis with anti-Shc.

View larger version (41K): [in this window] [in a new window]   Fig. 5.   Effects of JAK2 inhibition on the Shc tyrosine phosphorylation. A, cell lysates were immunoprecipitated with anti-Shc antibody and then probed with anti-phosphotyrosine (top) or Shc (bottom) antibody. Representative bands corresponding to the molecular mass of JAK2 (135 kDa) are shown from lysates from cells with Me2SO (DMSO) or with 60 µM AG-490 pretreatment for 16 h before H2O2 treatment (1 mM) (top). No difference in the amount of Shc was observed in lysates from any of the cell lines by Western blot analysis with anti-Shc (bottom). B, densitometric analysis of p52Shc tyrosine phosphorylation. Results were normalized by arbitrarily setting the densitometry of control cells (time = 0) to 1.0 (shown is mean ± S.D., n = 3).

We next investigated H₂O₂-mediated Shc tyrosine phosphorylation in Src/ and Fyn/ fibroblasts. In wild type fibroblasts, Shc tyrosine phosphorylation was maximally stimulated (5.31 ± 1.40-fold increase) by H₂O₂ at 5 min (Fig. 6, A and C). In Src/ fibroblasts, Shc tyrosine phosphorylation increased (maximum 5.90 ± 1.62-fold increase at 5 min) after H₂O₂ stimulation to an extent similar to wild type (Fig. 6, A and C). In contrast, in Fyn/ fibroblasts, H₂O₂-mediated Shc tyrosine phosphorylation was significantly inhibited (2.10 ± 1.2-fold increase at 5 min, p < 0.05, Fig. 6, B and C). These results indicate that H₂O₂-mediated Shc tyrosine phosphorylation is dependent, at least partially, on Fyn but not on c-Src in fibroblasts.

View larger version (34K): [in this window] [in a new window]   Fig. 6.   H2O2-mediated Shc tyrosine phosphorylation is inhibited in Fyn/ cells but not in c-Src/ cells. A, cells were stimulated for the indicated times with 1 mM H2O2, cell lysates were incubated with Shc antibody, and immunoprecipitates were analyzed by anti-phosphotyrosine and anti-Shc Western blotting. B, no difference in the amount of Shc was observed in lysates from any of the cell lines by Western blot analysis with anti-Shc (bottom). C, densitometric analysis of p52Shc tyrosine phosphorylation. Results were normalized by arbitrarily setting the densitometry of control cells (time = 0) to 1.0 (shown is mean ± S.D., n = 3). The asterisks represent significant differences compared with control (p < 0.05).

Restoration of JAK2 and Shc Responsiveness in Fyn/ Fibroblasts-- To provide further support for the role of Fyn in H₂O₂-mediated JAK2 activation and Shc tyrosine phosphorylation, Fyn/ fibroblasts were transiently transfected with B-Fyn or Src cDNA. As shown in Fig. 7A, there was no immunoreactive Fyn in cells transfected with empty vector and Src-transfected Fyn/ cells, whereas Fyn was restored in B-Fyn-transfected Fyn/ cells (Fig. 7A, upper). Likewise, there was no change in expression of Src in B-Fyn-transfected Fyn/ cells compared with empty vector-transfected cells (Fig. 7A, lower). There was a significant increase in expression of Src in Src-transfected Fyn/ cells compared with empty vector-transfected cells (Fig. 7A, lower). Cells transfected with B-Fyn responded to H₂O₂ with a two-fold increase in JAK2 (Fig. 7B) and Shc tyrosine phosphorylation (Fig. 7C) when compared with cells transfected with vector alone or Src. The two-fold increase in the JAK2 and Shc responses correlated well with the transfection efficiency, which ranged from 20-25% as judged from parallel transfection with a LacZ expression plasmid (data not shown). Thus, the JAK2 and Shc tyrosine phosphorylation responses to H₂O₂ in Fyn-deficient fibroblasts can be reconstituted by transfection with B-Fyn cDNA. Together, these results indicate that Fyn is an essential component in H₂O₂-mediated JAK2 and Shc tyrosine phosphorylation.

View larger version (43K): [in this window] [in a new window]   Fig. 7.   The induction of B-Fyn, but not Src, restores H2O2 responsiveness of JAK2 and Shc tyrosine phosphorylation in Fyn/ cells. Fyn/ cells were transfected with pME18S control vector, B-Fyn, or Src pME18S mammalian expression vector. A, pME18S empty vector, B-Fyn, or Src-transfected Fyn/ cells were harvested, and Western blot analysis was performed on whole cell lysates using anti-Fyn antibody (top) and anti-Src antibody (bottom). Cells were untreated or stimulated for 5 min with 1 mM H2O2. B, cell lysates were incubated with JAK2 antibody, and immunoprecipitates from each were analyzed by anti-phosphotyrosine and anti-JAK2 Western blotting. No difference in the amount of JAK2 was observed in lysates from any of the cell lines by Western blot analysis with anti-JAK2 (bottom). C, cell lysates were incubated with Shc antibody, and immunoprecipitates were analyzed by anti-phosphotyrosine and anti-Shc Western blotting. No difference in the amount of Shc was observed in lysates from any of the cell lines by Western blot analysis with anti-Shc (bottom).

H₂O₂ Stimulates Ras Activity via a JAK2-dependent Mechanism-- We next examined the effect of JAK2 inhibition on H₂O₂-mediated stimulation of Ras activation. Ras activation was assessed by monitoring the association between Ras-GTP and the RBD of Raf (21). Cell lysates of mouse fibroblasts stimulated with H₂O₂ were subjected to affinity precipitation with GST-RBD protein. The eluted proteins were then subjected to SDS-polyacrylamide gel electrophoresis and immunoblotted with anti-Ras antibody. Stimulation of fibroblasts with H₂O₂ caused maximal activation of Ras at 2 min as measured by GST-RBD association (Fig. 8A, lanes 5-7). AG-490 significantly attenuated Ras activity stimulated by H₂O₂ at 2 min (Fig. 8, A and B) (7.50 ± 1.51 versus 3.82 ± 0.81, p < 0.05). We also examined H₂O₂-mediated Ras activity in Fyn/ fibroblasts, and we found that its activity is inhibited in Fyn/ cells.² These data indicate that Fyn/JAK2 is involved in Shc tyrosine phosphorylation and Ras activation by H₂O₂.

View larger version (27K): [in this window] [in a new window]   Fig. 8.   Effect of JAK2 inhibition on Ras activation. A, cells were treated with Me2SO (DMSO) or 60 µM AG-490 for 16 h before timed exposures to H2O2 (1 mM). GTP-bound Ras was purified by affinity precipitation with a GST-RBD fusion protein followed by immunoblot analysis with anti-Ras antibody. There were no differences in the amount of the fusion protein (42 kDa) that was detected by Coomassie Brilliant Blue staining of the polyacrylamide gel, and no differences in the amount of Ras present in the cell extracts as determined by direct immunoblotting (data not shown). B, densitometric analysis of p52Shc tyrosine phosphorylation. Results were normalized by arbitrarily setting the densitometry of control cells (time = 0) to 1.0 (shown is mean ± S.D., n = 3). The asterisks represent significant differences compared with control (p < 0.05).

AG-490 Does not Completely Block ERK1/2 Activation by H₂O₂ but Alters the Time Course of Activation-- We next examined whether AG-490 blocked the ability of H₂O₂ to stimulate ERK1/2. ERK1/2 activity was measured with a phospho-specific ERK1/2 antibody that recognizes only the catalytically activated forms of ERK1/2. H₂O₂ activated ERK1/2 with peak at 5 min and return to basal level at 40 min (Fig. 9A). ERK1/2 activation at 5 min after H₂O₂ stimulation was blocked by AG-490 in a concentration-dependent manner (Fig. 9B), suggesting that JAK2 activation is necessary for ERK1/2 activation by H₂O₂ at 5 min. However, this inhibition was entirely transient in nature; maximal H₂O₂ activation of ERK1/2 occurred at 40 min and was similar in magnitude in AG-490-treated cells compared with control cells (Fig. 10, A and B). This delay in ERK1/2 activation was also found in Fyn/ cells; H₂O₂ activation of ERK1/2 at 40 min in Fyn/ cells was similar to ERK1/2 activation at 5 min in wild type cells (Fig. 10, C and D). These data indicate that inhibiting the H₂O₂-mediated pathway that requires Fyn and JAK2 (Fig. 11) does not prevent the activation of ERK1/2, but modifies its time course of activation. These findings suggest that there might be an alternative ROS-stimulated pathway responsible for the late phase activation of ERK1/2 in addition to Fyn/JAK2/Shc/Ras described in this study.

View larger version (23K): [in this window] [in a new window]   Fig. 9.   AG-490 inhibits ERK1/2 activation in a concentration-dependent manner in fibroblasts at 5 min after H2O2 stimulation. A, cells were stimulated for the indicated times with 1 mM H2O2, and ERK1/2 activity was measured by Western blot analysis with a phospho-specific ERK antibody. B, growth arrested fibroblasts were pretreated with Me2SO or the indicated concentrations of AG-490 for 16 h, and cells were lysed 5 min after H2O2 stimulation. ERK1/2 activity was measured by Western blot analysis with a phospho-specific ERK antibody (top). No difference in the amount of ERK1/2 was observed in lysates from any of the cell lines by Western blot analysis with anti-ERK1/2 (data not shown). Densitometric analysis of ERK1/2 activation (bottom). Results were normalized by arbitrarily setting the densitometry of control cells (time = 0) to 1.0 (shown is mean ± S.D., n = 3).

View larger version (27K): [in this window] [in a new window]   Fig. 10.   H2O2 activation of ERK1/2 is delayed by AG-490 pretreatment and Fyn/ cells. A, growth arrested fibroblasts were pretreated with Me2SO (DMSO) or AG-490 (60 µM) for 16 h, and cells were stimulated for the indicated times with 1 mM H2O2. ERK1/2 activity was measured by Western blot analysis with a phospho-specific ERK antibody. B, densitometric analysis of ERK1/2 phosphorylation. Results were normalized to control (time = 0), which was arbitrarily set to 1.0 (shown is mean ± S.D., n = 3). The asterisks represent significant differences compared with control (p < 0.05). C, cells were stimulated for the indicated times with 1 mM H2O2. Cells were harvested, and Western blot analysis was performed on whole cell lysates using anti-phospho-specific ERK1/2 antibody. No difference in the amount of ERK1/2 was observed in lysates from any of the cell lines by Western blot analysis with nonspecific anti-ERK1/2 (data not shown). D, densitometric analysis of ERK1/2 activity. Results were normalized by arbitrarily setting the densitometry of control cells (time = 0) to 1.0 (shown is mean ± S.D., n = 3). The asterisks represent significant differences compared with wild type cells (p < 0.05).

View larger version (11K): [in this window] [in a new window]   Fig. 11.   Model of H2O2-mediated signal transduction pathways to Ras and ERK1/2. Based on results with the specific JAK2 inhibitor AG-490, JAK2 regulates both Shc tyrosine phosphorylation and Ras activation by H2O2. Based on Fyn/ and Src/ cell experiments, Fyn (but not Src) regulates JAK2. Demonstration of a shift in H2O2-induced ERK1/2 activation by AG-490 and in Fyn/ cells suggests an alternative pathway for ERK1/2 activation, especially for the late phase of ERK1/2 activation.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The major findings of this study are first that H₂O₂ stimulates JAK2 in a Fyn-dependent and Src-independent manner, and second that JAK2 mediates phosphorylation of Shc and activation of Ras by H₂O₂. Redox-sensitive regulation of JAK2/Shc/Ras is thus a new function for Fyn. Data that support an essential role for Fyn and JAK2 in H₂O₂-mediated Shc/Ras activation include the following findings. 1) In Fyn/ fibroblasts, there was no JAK2 tyrosine phosphorylation in response to H₂O₂. In contrast, in Src/ fibroblasts, H₂O₂-mediated JAK2 tyrosine phosphorylation was not inhibited. 2) AG-490, a specific JAK2 inhibitor, prevented H₂O₂-mediated Shc tyrosine phosphorylation and Ras activation. 3) Likewise, Shc tyrosine phosphorylation and Ras activation by H₂O₂ were inhibited in Fyn/ fibroblasts, but not in Src/ fibroblasts. 4) Expression of Fyn, but not Src, in Fyn/ cells restored the response of Shc and JAK2 to H₂O₂. Our results are the first to show that Fyn, but not Src, is involved specifically in oxidative stress-mediated JAK2 activation, which also regulates a Shc/Ras signaling pathway.

Based on this study as well as previous work from our lab (6, 9) and other investigators (7, 25), we propose a scheme (Fig. 11) for ROS-mediated signal transduction leading to activation of Ras and ERK1/2. A novel aspect of this model is the specific role of Fyn, but not c-Src, to activate JAK2 and Ras. Previously we found that c-Src but not Fyn was required for H₂O₂-mediated BMK1/ERK5 activation in fibroblasts (9). In contrast, in this study, we found that Fyn but not c-Src was required for H₂O₂-mediated JAK2 activation in fibroblasts. These results indicate that c-Src and Fyn have separate roles in ROS-mediated signal transduction. Campbell et al. (17) have shown that the Janus kinases JAK1 and JAK2 (especially JAK1) are constitutively tyrosine phosphorylated in Src-transformed cells and suggested that JAK1, and possibly JAK2, are in an activated state in these cells. Furthermore, Uddin et al. (28) have shown that interferon- causes the SH2 domain of Fyn to interact with the activated form of interferon--dependent JAK2. Future studies will be required to define the precise nature of the downstream substrates for c-Src and Fyn.

In addition to activation of JAK2 by Fyn, this study demonstrates that H₂O₂-mediated Ras activation is partially dependent on Fyn and JAK2. Previous investigators have suggested an important relationship between two pathways used by cytokines and growth hormones to activate cells: the JAK2/STAT and the Ras/Raf/MEK/ERK pathway (14). A key convergence point of these pathways may be activation of Raf. Conversion of Ras to its GTP-bound form results in the binding of Raf to Ras, and this interaction with activated Ras localizes Raf to the plasma membrane and is often the first step in Raf activation. However, other investigators have reported that tyrosine kinases, including members of the Src kinase family and JAK2 phosphorylate Raf, thereby enhancing its activity. For example, Marrero et al. (24) reported that angiotensin II and platelet-derived growth factor induced JAK2·Raf complex formation, Raf-1 tyrosine phosphorylation, and ERK1/2 kinase activity, which were dependent on JAK2 activity. Based on this study, we propose that JAK2 is "upstream" of the adapter protein Shc and regulates H₂O₂-mediated Ras activation (Fig. 11).

The results for ERK1/2 activation suggest that H₂O₂ might activate two pathways, only one of which is dependent on Fyn and JAK2 (Fig. 11). In vascular smooth muscle cells, AG-490 inhibited the activation of ERK1/2 in response to either angiotensin II or platelet-derived growth factor (24). Aikawa et al. (10) reported that when Csk, a negative regulator of Src tyrosine kinases was overexpressed, activation of ERK1/2 by H₂O₂ at 10 min was abolished. We examined the role of JAK2 and Fyn in H₂O₂-mediated ERK1/2 activation with AG-490 treatment and in Fyn/ fibroblasts, and found that treatment with AG-490 or the absence of Fyn induced a temporal shift in activation of ERK1/2. However, the magnitude and duration of ERK1/2 activation were not altered. Of interest, McKenzie et al. (29) have reported that pretreatment with prostaglandin E and isobutylmethylxanthine to elevate cAMP attenuated the ability of growth factors to stimulate ERK1/2, when measured 5 min after growth factor stimulation. However, similar to the effect of AG-490 and the absence of Fyn this inhibition was not apparent at late times and the magnitude of ERK1/2 activation was not decreased. Recently, York et al. (30) have found that Rap1 mediates sustained ERK1/2 kinase activity induced by cAMP, so Rap1 could be a candidate to regulate this late phase activation of ERK1/2. Future studies will be required to determine the role of Rap1 in ROS-mediated signal transduction to ERK1/2.

In summary, we have shown that JAK2 is activated by ROS in a Fyn-dependent manner. The fact that H₂O₂-mediated activation of JAK2 required Fyn, but not c-Src, suggests that these two Src family kinases serve different intracellular functions with respect to oxidative stress. In addition, the demonstration that Fyn/JAK2 regulates H₂O₂-mediated Shc tyrosine phosphorylation and Ras activation, suggests that Fyn/JAK2/Shc/Ras signaling pathway may involve novel intracellular mediators.

	ACKNOWLEDGEMENTS

We thank Dr. D. Shalloway for providing the GST-RBD construct and Drs. H. Umemori and Tadashi Yamamoto for providing B-Fyn and Src cDNA construct. We also thank Drs. C. Yan, H. Ueba, M. Okuda, B. Gallis, and G. Daum for their invaluable assistance and critical reading of this manuscript.

	FOOTNOTES

^* This work was supported by a grant from the Japanese Heart Foundation and Bayer Yakuhin Research Grant Abroad (to J. A.), and Grants HL44721 and HL49192 from National Institutes of Health (to B. C. B.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Cardiology Unit, Box 679, 601 Elmwood Ave., University of Rochester School of Medicine and Dentistry, Rochester, NY 14642. Tel.: 716-273-1947; Fax: 716-473-1573; E-mail: bradford_berk@urmc.rochester.edu.

² J. Abe and B. C. Berk, submitted.

	ABBREVIATIONS

The abbreviations used are: ROS, reactive oxygen species; ERK, extracellular signal-regulated kinase; JAK, Janus kinase; RBD, Raf-binding domain; GST, glutathione S-transferase.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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